Stabilized aqueous enzyme compositions

ABSTRACT

Aqueous amylolytic enzyme-containing compositions comprising water, amylolytic enzyme, a water-soluble calcium salt, an organic co-stabilizing agent selected from aliphatic glycols and 1,3-propanediol and, optionally, a nonionic or zwitterionic detergent are disclosed. The compositions, useful as starchdegrading compositions, are stabilized substantially against loss of amylolytic enzyme activity during storage.

United States Patent [19] Berry 5 STABILIZED AQUEOUS ENZYME COMPOSITIONS[75] Inventor: Jim S. Berry, Springfield Twsp.,

Hamilton County, Ohio [73] Assignee: The'Procter & Gamble Company,Cincinnati, Ohio [22 Filed: Mar. 6, 1972 [21] Appl. No.: 232,300

Related U.S. Application Data I [63] Continuation of Ser. No. 786,432,Dec. 23, 1968,

abandoned.

[52] U.S. Cl 252/l53,,195/63, 252/132, 252/544, 252/545, 252/D1G. 1,252/D1G. l2, 252/D1G. 14, 252/546, 252/547 [51] Int. Cl. C07g 7/02, C11d 7/42, C1 1d 7/50 [58] Field of Search 195/63, 68; 424/94; 252/89,132, 135, 546, 398, 403, DIG. 12, 153

[56] References Cited UNITED STATES PATENTS 2,164,914 7/1939 Gore 195/682,164,936 7/1939 Miller et al. 195/68 2,594,356 4/1952 Schwimmer et a1.

11] 3,819,528 [45.1 June 25, 1974 3,415,804 12/1968 Polson 424/94 X3,451,935 6/1969 Roald et al. 252/135 3,457,175 7/1969 Curry 252/893,472,783 10/1969 Smillie 252/89 3,524,798 8/1970 Lloyd et al 195/31 RFOREIGN PATENTS OR APPLICATIONS 37-16,696 10/1962 Japan OTHERPUBLICATIONS Primary Examiner-Herbert B. Guynn Assistant Examiner-DennisL. Albrecht Attorney, Agent, or FirmRichard C. Witte; George- W. Allen;Julius P. Filcik [57] ABSTRACT Aqueous amylolytic enzyme-containingcompositions comprising water, amylolytic enzyme, a water-solublecalcium salt, an organic co-stabilizing agent selected from aliphaticglycols and 1,3-propanediol and, optionally, a nonionic or zwitterionicdetergent are disclosed. The compositions, useful as starch-degradingcompositions, are stabilized substantially against loss of amylolyticenzyme activity during storage.

19 Claims, No Drawings STABILIZED AQUEOUS ENZYME COMPOSITIONS This is acontinuation of application Ser. No.

, 786,432, filed Dec. 23, 1968, now abandoned.

FTELD OF THE INVENTION of starchy materials and characterized bystabilization against loss of amylolytic activity.

The use of amylolytic enzymes in the alteration and- /or degradation ofstarchy materials is known. For example, US. Pat. No. 2,607,359 (Aug.19,1962) describes compositions containing an amylolytic enzyme usefulin facilitating the removal of porous materials such as wallpapers,labels and casein type pastes from surfaces to which the porousmaterials are held by a starch-containing adhesive. Similarly, Jaag inSeifen, Ole, Fette, Wachse 88, No. 24, pp. 789-793, (Nov. 1962)describes the use of amylolytic enzymes in laundry formulations. Theseenzymes aid in the laundry process by attacking starchy soils and stainsfound on soiled fabrics and decomposing and/or altering them so as torender them more removable during laundering.

Enzymatic materials are expensive and powerful materials which must bejudiciously formulated and used. These enzymes when employed in aqueouscompositions are unstable and suffer appreciable destruction during longperiods of storage as evidenced by substantial loss in starch-degradingand/or soiland stainremoving efficacy. The loss in amylolytic activityis particularly severe under conditions of high temperature.Furthermore, aqueous laundering solutions containing amylolytic enzymesoften contain additional components desirable in the laundering processbut which have an adverse effect on the amylolytic enzyme. Proteolyticenzymes, for example, while useful in providing proteinaceous soilandstain-removing properties in laundering compositions, often tend to havesuch an adverse degrading effect on amylolytic enzymes.

Attempts have been made in the art to provide amylolyticenzyme-containing compositions wherein the enzymatic activity ispreserved by the incorporation of a stabilizing agent. These attemptshave generally involved incorporation in such compositions ofwatersoluble calcium salts. A trade bulletin describing bacterialamylases derived from Bacillus subtilis, published by Daiwa Kasei K. K.of Osaka, Japan, describes the stabilization of bacterial amylase withcalcium and sodium ions. Similarly, l-lamada et al., Agr. Biol. Chem,31, No. 1, pp. 1-6 (1967), describe the stabilizing effect of calciumions'on oz-amylase. The employment of calcium salts to impede loss ofamylolytic activity, particularly at high temperatures, has not beenentirely satisfactory, particularly over extended periods of storage athigh temperatures. Accordingly, there has been a need for amylolyticenzyme-containing compositions having improved amylolytic enzymestability.

It is therefore an object of this invention to provide stabilizedaqueous amylolytic enzyme-containing compositions which retainsubstantially their amylolytic activity upon storage.

lt is another object of this invention to provide aqueous amylolyticenzyme-containing compositions stabilized substantially against loss ofactivity by the presence of minor amounts of enzyme-stabilizingcompounds.

Other objects of this invention will be obvious from consideration ofthe invention which is more fully described hereinafter.

SUMMARY OF THE INVENTION These and other objects of the presentinvention re achieved by the provision of aqueous amylolyticenzyme-containing compositions containing minor amounts of calcium ionand certain organic costabilizing agents. The aqueous compositions ofthe present invention can additionally contain nonionic or zwitterionicdetergent components to enhance the stability of the amylolytic enzymesin the aqueous compositions of this invention and to enhance thedetergent properties of these compositions. The present invention isbased in part on the discovery that extended periods of stabilizationcan be achieved by introducing into aqueous enzyme-containingcompositions a source of calcium ion and an organic compound selectedfrom the group consisting of aliphatic glycols and 1,3- propane diol.

The stabilized aqueous enzyme-containing compositions of this inventioncomprise 1. from about to about 97 percent water;

2. from about 0.001 to about 1 percent amylolytic enzyme;

3. from about 0.001 to about 1 percent with respect to calcium ion of awater-soluble enzymestabilizing calcium salt;

4. from about 2 to about 27 percent of an organic compound selected fromthe group consisting of aliphatic glycols having the formula wherein xis from about 1 to about 200; and 1,3- propane diol; and

5. from 0 to about 15 percent of a detergent selected from the groupconsisting of nonionic detergents and zwitterionic detergents.

The amylolytic enzymes which can be stabilized in aqueous solution bythe action of the hereinbefore described combination of calcium ion andorganic compound are known materials and can be of fungal, plant, animalor bacterial-origin. Suitable amylolytic enzymes include the a-amylaseswhich are particularly well suited for breaking down starch molecules asthey attack the a -glycosidic linkages in starch. The degraded shortchains are easily removed from their environment with water or aqueoussolutions of detergents. Examples of suitable amylolytic enzymes includethe a-amylases of mold origin including those derived from Aspergillusoryzae, Aspergillus niger, Aspergillus alliaceus, Aspergillus wentii,and Pencillium glaucum. The

' a-amylases derived from cereal grains, pancreatic sources and suchbacteria as Bacillus subtilis Bacillus macerans, Bacillus mesentericusand Bacillus thermophilus are also useful herein. These enzymes areactive in the pH range of from about 4.5 to about 10 and at temperaturesfrom about 60F. to about F. Optimum activity of these a-amylases isgenerally exhibited in the pH range of from about 5.5 to about 7.5.

Preferred amylolytic enzymes herein are the a-amylases derived from thebacterial organism Bacillus subtilis. These amylases provide excellentdesizing and starch digestive properties and are especially useful inthe laundering of textile materials containing soils and stains of astarchy nature.

The amylolytic enzymes useful herein can be employed in a pure state.Generally they are employed in the form of a powdered commerciallyavailable preparation wherein the amylolytic enzyme is present in anamount of from about 2 to about 80 percent of the preparation. Theremaining portion, i.e. about to about 98 percent, comprises inertvehicle such as sodium sulfate, calcium sulfate, sodium chloride, clayor the like. In preparing the stabilized aqueous starchdegradingcompositions of this invention, such commercial enzyme preparations areadmixed with water and the remaining components of the compositions. Theactive enzyme content of these commercial enzyme compositions is theresult of manufacturing methods employed and is not critical herein solong as the finished compositions of this invention have the hereinafterspecified enzyme content. Insoluble inert materiby methods known in theart so long as the stabilized compositions of the invention provide anamount of amylolytic enzyme activity sufficient to provide desirablelevels of starch degrading properties.

As used herein, amylolytic activity refers to the tendency of anamylolytic enzyme to perform the desired function of catalyticalteration and/or degradation of starchy materials Stability, as usedherein, refersto the tendency of an amylolytic enzyme to retain itsenzymatic activity. The activity level of amylolytic enzyme suitableherein can be determined by-numerous methals are generally removed fromthe compositions of this invention to provide aqueous compositions whichare clear and substantially free of precipitated deposits. Specificexamples of commercial enzyme preparations suitable for use herein andthe manufacturers thereof include: Diasmen c r-amylase (Daiwa Kasei K.K. To kyo, Japan); Rapidase a-amylase' THC- (Rapidase, Seclin, France);Novo Bacterial a-amylase (Novo Industri, Copenhagen, Denmark);Wallerstein a-amylase (Wallerstein Company, Staten Island, New York);Rhozyme-33 and Rhozyme H-39 (Rohm & Haas, Philadelphia, Pennsylvania).

Preferred herein is a powdered enzyme preparation containing a-amylaseand amixture of alkaline and neutral proteases available as CRD-Protease(or Monsanto DA-lO) from Monsanto Company, St. Louis, Missouri. Thiscomposition contains about 3 a-amylase and is useful herein to providethe compositions of the invention with amylolytic and proteolytic enzymeactivity. Mixtures of proteases and 'a-amylases are preferred herein andinclude the enzyme preparations described in US. Pat. No, 3,031,380 toMinagawa et al. (Apr. 24, 1962).

The amount of amylolytic enzyme employed'in the compositions of thisinvention can vary depending.

upon the activity of the enzyme or enzyme preparation, conditions of pHand the intended use of the compositions. When the stabilized aqueouscompositions of this invention are employed as spot removers, theyshould contain an amount of amylolytic enzyme sufficient to remove thestarchy soils and stains normally encountered in a laundering situation.Normally the compositions of this invention are prepared to contain fromabout 0.001 to about l-percent enzyme by weight of the aqueouscomposition on a pure enzyme basis. For best results, the compositionspreferably contain from about 0.01 to about 0.5 percent amylolyticenzyme. When a commercially available powdered enzyme preparation isemployed as the source of amylolytic enzyme, the compositions of thisinvention contain from about 0.1 to about 4.0 percent of the powderedamylo lytic enzyme-containing preparation as it is available incommercial form e.g., containing from' about 2 to about 80 percentactive enzyme. It will be appreciated that when such preparations areemployed herein, the amount of the preparation required to provideaqueous compositions having desirable levels of amylolytic activity willdepend on the activity level of the enzymecontaining preparationemployed. The precise amounts of such materials employed can be readilydetermined ods. Asuitable method is the 3,5-dinitrosalicylate assaymethod. In accordance with this method, a sample of amylase is allowedto catalyze the hydrolysis of the 1,4- a-glycosidic bonds of starch andglycogens for five minutes at a temperature of 37C. at a pH of 6.0. Thereaction is terminated by the addition of buffered sodium3,5-dinitrosalicylate, the color is developed and the amount of maltosedetermined by spectrophotometric response and comparison with solutionsof analytical grade maltose hydrate. The amylase has one activity unitfor each 0.4 mg. of maltose hydrate produced during hydrolysis under thespecified conditions. The amylase activity method is well known and isdescribed with particularly in P. Bemfeld, Methods in Enzymol. Vol I. p.149 (1955).

As hereinbefore described, the present invention is based in'part uponthe surprising discovery that extended periodsof enzyme stabilizationcan be achieved by incorporating in'toaqueous enzyme solutionsacombination of water-soluble calcium salt and organic co- 7 stabilizingcompound hereinbefore described. The water-soluble salts of calciuminclude, for example, calcium chloride, calcium acetate, calciumcitrate, calcium glycerol phosphate, calcium gluconate, calciumglucoheptanate, calcium lactate, calcium levulinate, calciumlactobionate, calcium malate, calcium lactophosphate, calcium succinate,calcium maleate, and calcium sulfate. The stabilized compositions of theinvention are-prepared to contain from about 0.001 to about l percent ofthe stabilized composition with respect to the calcium ion. Preferablyfrom about 0.005 to about 0.05 percent with respect to the calcium ion,

isemployed for best stabilization. As described hereinbefore, certain ofthe commercially available enzyme I preparations suitable hereincontain, in addition to active enzyme, certain inert materialsincludingfor example, calcium chloride or calcium sulfate. When such an enzymepreparation is employed as the source of amy-' lolytic enzyme, an amountof calcium ion is also incorporated thereby.v Additional calcium ion isconveniently provided by the addition of one or more of the calciumsalts hereinbefore described so as to provide a level of calcium ionwithin'the hereinbefore described range. Preferred calcium salts includecalcium acetate,

' calcium sulfate and calcium chloride. i

The organic co-stabilizers which in concert with cal cium ion provideenhanced amylolytic enzyme activity include the aliphatic glycols andl,3-propanediol. The aliphatic glycols employed herein have the formulanorcn cn o' a wherein x-is from 1 to about 200, and include ethyleneglycol and the polyethylene glycols. The polyethylene glycols usefulherein are those wherein'x in the hereinbefore described formula rangesfrom 2 to about 200 andv include diethylene glycol, triethylene glycoland the corresponding polymers of ethylene oxide wherein the averagenumber of oxyethylene groups ranges upward from 4 (tetraethylene glycol)to about 200.

The aliphatic glycols useful herein range in consistency from lightliquids to white waxy solids and dissolve in water to form clearsolutions. Preferred aliphatic glycols herein include diethylene glycoland triethylene glycol. Also preferred are the polyethylene glycolswherein the average value of x is from 4 to about 80. These polyethyleneglycols have average molecular weights of about 200 to about 3500 andare commercially available under the trade designation Carbowax withnumerical designation referring to average molecular weight, e.g. 200,400, 600, 1000, or the like. The upward numerical gradation correspondsto increasing molecular weight, increasing melting point and decreasingwater-solubility. Mixtures of aliphatic glycols of the invention can beemployed herein.

It has been found that 1,3-propanediol also provides an amylolyticenzyme-stabilizing effect as hereinbefore described. This compound is apreferred co-s'tabilizing agent herein and provides excellentstabilizing effects.

The organic amylolytic enzyme co-stabilizing compounds of this inventionare employed in minor but effective' amounts ranging from about 2 toabout 27 percent by weight of the composition. Preferably the stabilizedcompositions are prepared to contain from about 5 to about percent byweight of the co-stabilizing agent. The latter range is preferred fromthe standpoint of optimum stabilizing effects, particularly over longstorage periods at high temperatures.

While the mechanism by which the calcium salts and organicco-stabilizing agent hereinbefore described coact to protect amylolyticenzymes against loss of activity is not precisely known, the combinationof salt and organic compound provides levels of enzyme stabilitysubstantially greater than can be achieved by conventional calciumstabilization alone. This stabilization effect is observed even in thepresence of proteases which tend to exert a harmful denaturing effect ona-amylases.

Water-soluble nonionic and zwitterionic detergents can be employed, asoptional ingredients, in the compositions of this invention. Thesedetergents enhance considerably the storage stability of the amylolyticen- 1 zymes employed herein and significantly improve the detergentcharacteristics of the composition. Because of these usefulcharacteristics it is preferred to include nonionic and zwitterionicdetergents in the aqueous en zyme compositions of the invention. Thenonionics and zwitterionics can be utilizedherein in amounts rangingfrom 0 to about 15 percent, and preferably from 4 to 10 percent, byweight of the enzyme-containing composition.

Examples of suitable nonionics for use herein include:

l. The polyethylene oxide condensates of alkyl phenols, e.g., thecondensation products of alkyl phenols having an alkyl group containingfrom about six to 12 carbon atoms in either a straight chain or branchedchain configuration with ethylene oxide, the said ethylene oxide beingpresent in amounts equal to 5 to moles of ethylene oxide per mole ofalkyl phenol. The alkyl substituent in such compounds may be derivedfrom polymerized propylene, diisobutylene, octene or nonene, forexample. 2. Those nonionic synthetic detergents derived from thecondensation of ethylene oxide with the product resulting from thereaction of propylene oxideand ethylene diamine. For example, compoundscontaining from about 40 to about per cent polyoxyethylene by weight andhaving a molecular weight of from about 5,000 to about 11,000 resultingfrom the reaction of ethylene oxide groups with a hydrophobic baseconstituted of the reactionproduct of ethylene diamine and excesspropylene oxide, said base having a molecular weight of the order of2,500 to 3,000 are satisfactory. 3. The condensation product of 1 moleof aliphatic alcohols having from eight to 22 carbon atoms, in eitherstraight chain or branched chain configuration, with from 5 to 40 molesof ethylene oxide, e.g., a coconut alcohol-ethyleneoxide'condensate-having from 5 to 40 moles of ethylene oxide per mole ofcoconut alcohol, the coconut alcohol fraction having from 10 to 14carbon atoms. 4. The unsubstituted amides and the monoethanol anddiethanol amides of fatty acid having acyl moieties of from about eightto about 22 carbon atoms. These acyl moieties are normally derived fromnaturally occurring glycerides (e.g., coconut oil, palm oil, soybean oiland tallow), but can be derived synthetically (e.g., by the oxidation'ofpetroleum, or by hydrogenation of carbon monoxide by the Fischer-Tropschprocess). 5. Long chain tertiary amine oxides corresponding to thefollowing general formula RRIRIIP V,

wherein R is an, alkyl, alkenyl or monohydroxyalkyl radical ranging from10 to 22 carbon atoms in chain length and R and R" are each alkyl .ormonohydroxyalkyl groups containing from one to three carbon atoms. Thearrow in the formula is a conventional representation of a semi-polarbond. Examples of suitable phosphine oxides are found in US. Pat. No.3,304,263 which issued Feb. 14, 1967, and include:dimethyldodecylphosphine oxide and bis-( 2-hydroxyethyl)-dodecylphosphine oxide.

7. Long chain sulfoxides having the formula wherein R is an alkylradical containing from about 10 to about 22 carbon atoms, from 0 toabout 5 ether linkages and from 0 to about 2 hydroxyl substituents, atleast one moiety of R being uninterrupted by ether linkages andcontaining from about 10 to about 18 car- 7 straight chain or branched,and wherein one of the aliphatic substituents contains from about eightto 22 carbon atoms and one contains an anionic water solubilizing-group,e.g., carboxy, sulfo, sulfato, phosphate or phosphono. Examples ofcompounds falling within this definition are 3-(N,N-dimethyl-N-hexadecylammonio) propane-l-sulfonate and3-(N,N-dimethyl-N- hexadecylammonio )-2-hydroxypropanel-sulfonate. Formore examples of zwitterionic synthetic detergents, see Diehl and Smith,Laundering Fabrics in Cold Water Containing a Synthetic DetergentComposition, Canadian Patent No. 708,147 issued Apr. 20, 1965 at page 6,lines 1 22. This disclosure is specifically incorporated herein byreference.

Mixtures of various nonionic detergents or mixtures of nonionicdetergents and zwitterionic detergents can be employed. Preferred hereinare the condensation products of 1 mole of aliphatic alcohol havingeight to 22 carbon atoms with from 5 to 40 moles of ethylene oxide, e.g.tallow alcohol ethoxylated with 11 or 30 moles of ethylene oxide andcoconut alcohol ethoxylated with 6 moles of ethylene oxide. Alsopreferred are the 3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-l-sulfonates wherein the alkyl has from eight to 22carbon atoms, e.g. 3-(N,N-dimethyl- N-coconutalkylammonio)-2-hydroxypropane- 1- sulfonateand the 3-(N,N-dimethyl-N-alkylammonio)propane-l-sulfonates wherein the alkyl has from eight to 22 carbonatoms, e.g. 3-(N,N-dimethyl N- tallowalkylammonio) propane- 1-sulfonate. These compounds in addition to providing amylase stabilityper se enhance the stabilization of calcium and organic costabilizingcompound. In addition they provide excellent detergency properties.

The stable compositions of the present invention are prepared to containfrom about 65 to about 97 percent by weight of water. Preferably fromabout 72 to about 95 percent is employed. Demineralized water ispreferred, although not mandatory for use herein.

The various components of the enzyme compositions of this invention canbe mixed together in any order. However, it is preferred that astabilizer-water mixture be prepared first and the enzymes added theretoto prevent any degradation or deactivation which might occur by addingthe enzyme to water which does not contain the enzyme-stabilizingcombination of the invention. The optional detergent components can beadded at any time.-

The pH of the stabilized aqueous enzyme compositions of this inventiongenerally ranges from about 5.0 to 10.0 and preferably ranges from about6.5 to about 8.5. Maximum stabilizing affects are obtained in thepreferred pH range. The pH can be raised with a base, e.g., sodium orpotassium hydroxide, or lowered with an acid, e.g., hydrochloric acid.

It is also preferred, although not mandatory, that a preservative beadded to the compositions to prevent bacterial and fungal growth. Phenylmercuric acetate which is generally utilized herein in amounts rangingfrom about 10 to about 40 parts per million of the compositions is aneffective preservative. Any preservative compatible with the componentsof the compositions can be utilized herein.

The stabilized aqueous compositions of this invention can also containany of the usual detergent adjuvents, diluents and additives so long asthey do not substantially interfere with the activity of the enzymaticcomponents. For example, perfumes, anti-tamishing agents, inert saltssuch as sodium sulfate, anti-redeposition agents, bacten'ostatic agents,dyes, fluorescers, suds builders, suds depressors, and the like, can beutilized herein without detracting from the advantageous prop ertiesof'these compositions. It is preferred that the compositions of thepresent invention contain in addition certain proteolytic enzymes. Theseenzymes include the alkaline proteases, neutral proteases, and acidproteases which aid materially the removal of proteinaceous soils andstains from laundered textiles. The employment of proteolytic enzymes incombination with the amylolytic enzymes of the present invention ispreferred from the standpoint of facilitating the removal of a broadspectrum of varied soils and stains. The preferred proteolytic enzymesare the subtilisins, obtained from the bacterial organism, Bacillussubtilis. When proteolytic enzymes are included in the compositions ofthe present invention, it is desirable to include any of the knownproteolytic enzyme-stabilizing materials known in the art to therebyenhance proteolytic enzyme activity upon storage. Suitable proteolyticenzyme stabilizing materials are described for example in Ser. No.683,196, entitled Stabilized Aqueous Enzyme Preparation filed Nov. 15,1967 by Charles Bruce McCarty.

The compositions of this invention can be employed as spot removers,detergent additives'or as detergent cleaning compositions per se.Thesecompositions can EXAMPLES The following examples merely serve toillustrate the invention in specific detail and when read in conjunctionwith the foregoing description will aid in determining the full scope ofthe present invention. The examples are merely illustrative and are notintended to restrict this invention. All parts, percentages and ratiosset forth herein are by weight unless otherwise indicated.

The following compositions were prepared and stored in closed glassbottles for the lengths of time indicated in Table I. Each compositioncontainined 10 percent by weight of the organic co-stabilizing agent; 1percent Monsanto CRD-Protease (a commercially available mixture ofproteases and amylases derived from Bacillus subtilis); and 89 percentof an aqueous stock solution containing 0.01 percent calcium acetatemonohydrate and 0.29 percent sodium chloride. The amylolytic activity ofeach composition was measured at the stated intervals by the assaymethod hereinbefore described.

Control samples stored under identical conditions were also evaluatedfor retention of enzymatic activity. In Control-1, no organicco-stabilizing agent was employed. In the case of Control-2, no organiccostabilizing agent was present and thestock solution was replaced withdistilled water, i.e., no calcium acetate monohydrate or sodium chloridewas present. The results are tabulated as follows:

ionic or zwitterionic was added and the enzyme was added last. Theenzymes employed were Alcalase (a proteolytic enzyme preparation havingacrystalline enzyme content of about 6 percent and derived from Bacillussubtilis); and/or Monsanto CRD-Protease (a mixture of proteolytic andamylolytic enzymes derivedv from Bacillus subtilis). Ethanol, wherepresent, was employed as a stabilizer for the proteolytic enzyme. Ineach example, an aqueous stock solution, hereinbefore described, wasemployed in an amount to bring the balance of the composition to 100percent. The composi- 'tions were stored for the periods of timeindicated in Table II at a temperature of 100F. and their amyloly- Thefollowing stabilized compositions, Examples 4to 29, were prepared. Ineach example, the water (containing calcium acetate monohydrate andsodium chloride) organic co-st'abilizing agent and ethanol (lwhereemployed) were thoroughly mixed, the nontic activity evaluated ashereinbefore described. The

compositions of Examples 4 to 29 perform well as spot removers, asadditives to detergent compositions and as'laundry detergents per se.

Table II Remaining Enzyme After Storage v at 100F. for

Mon- 7c Alcasanto Surfactant* Weeks Ex. Ca Ethanol Organic Co-stabilizerlase CRD A B D E 2 4 6 8 4 0.0017 10 Diethylene Glycol (10%) 0.5 0.5 10088 100 100 5 0.0019 5 Diethylene Glycol (5%) 0.5 0.5 5 100 '79 50 60.0017 10 Triethylene Glycol (10%) 0.5 0.5 5 100 83 58 63 7 0.0019 5Triethylene Glycol (5%) 0.5 I 0.5 5 98 53 30 8 0.0017 10 PolyethyleneGlycol 380 (10%) 0.5 0.5 5 I 83 45 33 9 0.0019 5 Polyethylene Glycol 380(5%) 0.5 0.5 5 82 61 31 11 10 0.0017 10 Polyethylene Glycol 4000 (10%)0.5 0.5 5 70 42 18 7 1 I 0.0019 5 Polyethylene Glycol 4000 (5%) 0.5 0.55 79 32 18 4 12 0.0020 Triethylene Glycol 10%) 0.5 0.5 90 100 100 100 130.0020 Polyethylene Glycol 4000 10%) 0.5 0.5 78 87 83 82 14 0.0019 10Diethylene Glycol (10%) 0.5 0.5 5 68 75 59 59 15 0.0017 10 DiethyleneGlycol (10%) 0.5 0.5 5 77 45 54 16 0.0017 10 Triethylene Glycol (10%)0.5 0.5 5 85, 75 74 17 0.0017 10 Polyethylene Glycol 380 (10%) 0.5 0.5 59 1 73 64 65 18 0.0017 10 Polyethylene Glycol 4000 (10%) 0.5 0.5 5 87 6957 64 19 0.0020 Diethylene Glycol (10%) 0.5 0.5 96 73 73 75 20 0.0018Diethylene Glycol (10%) 1.0 10 95 89 21 0.0018 Diethylene Glycol (10%)1.0 10 87 72 22 0.0018 Diethylene Glycol (10%) 1.0 10 59 49 23 0.0018Diethylene Glycol (10%) 4 1.0 l0 51 36 24 0.0018 Diethylene Glycol (10%)1.0 10 48 30 25 0.0018 1.3-Propanediol (10%) 1.0 10 89 '81 26 0.00181,3-Propanediol (10%) 1.0 l0 77 72 27 0.0018 1.3-Propanediol (10%) 1.010 86 65 28 0.0018 1,3-Propanediol (10%) 1.0 10. 100 55- 29 0.00181,3-Propanediol (10%) 1.0 10 1'00 74 ATallow alcohol ethoxylated with l1 moles of ethylene oxide. 'BTallow alcohol cihoxylated with 30 moles ofethylene oxide. C-Coconut alcohol ethoxylated with 6 moles of ethyleneoxide.

D- HAPS 3(N.N-dimelhyl-N-alkylammonio)Z-hydroxy propanc-l-sulfonatewherein the alkyl group is derived from middle-cut coconut alcohol: 2%cm; 66% C 23'; C and 9; C 'E3-( N.N-dimethyl-N-tallowalkylammonio)propane-l-sulfonute.

EXAMPLE 30 A stabilized aqueous enzyme composition is formu-v latedaccording to this invention from the following components:

Thiscomposition can be employed without dilution V as a soilandstain-removing composition to remove or facilitate removal of starchyand proteinaceous matter from textile materials. The soilandstain-removing efficacy is demonstrated even after extended periods ofstorage (8 weeks) at elevated temperature (120F.).

The composition of this example can be employed as an additive tocommercial detergent formulations. When about 1.2 ml. of the compositionis added per gallon of washing solution, excellent soiland stain--removing properties are demonstrated.

EXAMPLE 31 I Similar results areobtained when the following or-' amountof calcium ion in that stabilization of amyloganic co-stabilizingcompounds are employed in lieu of the co-stabilizing compounds employedin. Examples 1 to 29 in-that the amylolytic enzyme is stabilized inaqueous solution: ethylene glycoljdiethylene glycol; triethylene glycol;tetraethylene glycol; polyethylene glycol 200; polyethylene glycol 300;polyethylene glyc'ol 380; polyethylene glycol 600; polyethylene glycol1000; polyethylene glycol 1500; polyethylene glycol 4000; polyethyleneglycol 6000; and 1,3-pr0panediol.

Similar results are obtained when the following amylolytic enzymes areemployed in lieu of those employed in Examples 1 to 29 in that thea-amylase is stabilized in aqueous solution: Diasmen a-amylase; Rapidasea-amylase THC-25; Novo Bacterial a-amylase; Wallerstein a-amylase;Rhozyme-33 and Rhozyme H-39.

Similar results are-obtained when the following nonionic andzwitterionic detergents are substituted for the tallow alcoholethoxylates, coconut alcohol ethoxylate,3-(N,N-dimethyl-N-middlecut-coconutalkylammonio)Z-hydroxypropane-l-sulfonateand 3- (N,N-dimethyl-N-tallowalkylammonio) propanel sulfonate employedin Examples 4 to l 1, 14 to 18 and 20 to 29 in that the stabilization bycalcium ion and organic co-stabilizing compound is enhanced andexcellent cleaning properties are provided: decyl phenol ethoxyl'atedwith 20 moles of ethylene oxide per mole of decyl phenol, hexadecanoicamide, hexadecanoic diethanol amide, dimethyldodecylamine oxide,dimethyldodecylphosphine oxide, and dodecyl methyl sulfoxide, thecondensation product of ethylene oxide with the condensation product ofpropylene oxide with propylene glycol, the ethylene oxide portion of thecompound being 50 percent of the total weight of the compound andthetotal molecular weight of the compound being about l700;-thecondensation product of ethylene oxide with the condensation product ofpropylene oxide .and ethylene diamine wherein the product contains about65 percent polyethylene oxide by weight and the total molecular weightof the compound is 6000.

Similar results are obtained when the calcium acetate monohydrate ofExamples 1 to 29 is replaced with the following calcium salts in amountsproviding an equal lytic enzyme in aqueous solution is observed: calciumchloride; calcium citrate; calcium glycerol phosphate; calcium'gluconate; calcium glucoheptanate; calcium lactate; calcium levulinate;calcium lactobionate; calcium malate; calcium lactophosphate; calciumsuccinate; calcium maleate; and calcium sulfate.

What is claimed is:

1. A stabilized aqueous enzyme composition consisting essentially of byweight of the composition:

1. from about 65 percent to about 97 percent water; I

2. from about 0.001 percent to about 1 percent amylolytic enzyme; 3.from about 0.001 percent to about 1 percent with respect to calcium ionof a water-soluble enzymestabilizing calcium salt;

4. from about 2 percent to about 27 percent of an organic co-stabilizingcompound selected from the group consisting of aliphatic glycols havingthe formula HO CH CH OhH wherein x is from about 2 to about 200; and1,3- propanediol; and 5. from 0 to about 15% of a detergent selectedfrom the group consisting of nonionic detergents and zwitterionicdetergents. 2. The composition of claim I wherein the costabilizingcompound is an aliphatic alcohol having the formula wherein is fromabout 2 to about 200; and wherein the amylolytic enzyme is an a-amylasecharacterized by amylolytic activity in the pH range of from about 4.5to about'l0 and at a temperature of from about costabilizing compound isselected from the group consisting of diethylene glycol; triethyleneglycol; and glycols of the formula wherein the average value of x isfrom 4 to about 80.

7. The composition of claim 6 wherein from about 4 to about 10 percentof a nonionic or zwitterionic detergent is present.

8. The composition of claim 7 wherein the detergent is selected from thegroup consisting of condensation products of 1 mole of aliphatic alcoholhaving from eight to 22 carbon atoms with from 5 to 40 moles of ethyleneoxide; 3-(N,N-dimethyl-N-alkylammonio) propanel-sulfonate wherein thealkyl has from eight to 22 carbon atoms; and 3-(N,N-dimethyl-N- r 13alkylammonio )-2-hydroxypropane- 1 -sulfonate wherein the alkyl has fromeight to 22 carbon atoms. 5

9. The composition of claim 8 wherein the detergent is3-(N,N-dimethyl-N-coconutalkylammonio)-2- hydroxypropane-l-sulfonate andthe organic costabilizing compound is triethylene glycol.

10. The composition of claim 8 wherein the calcium salt is selected fromthe group consisting of calcium acetate, calcium sulfate and calciumchloride.

11. A stabilized aqueous enzyme composition consisting essentially of byweight of the composition:

1. from about 65 to about 97 percent water;

2. from about0.00l to about 1 percent amylolytic enzyme;

3. from about 0.001 to about 1 percent with respect to caclium ion of awater-soluble, enzymestabilizing calcium salt;

4. from about 2 to about 27 percent of a 1,3-

propanediol co-stabilizing compound; and

5. from 0 to about 15 percent of a detergentselected from the groupconsisting of nonionic detergents and zwitterionic detergents.

12. The composition of claim 11 wherein the amylolytic enzyme is ana-amylase characterised by amylolytic activity in the pH range of fromabout 4.5 to about and at a temperature of from about 60F. to about150F.

13. The Composition of claim 12 wherein the a-amylase is derived fromBacillus subtilis.

14. The composition of claim 13 wherein the a-amylase is present in anamount of from about 0.01 to about 0.5 percent.

15. The composition of claim 14 wherein the calcium ion is present in anamount of from about 0.005 to 0.05 percent and the organicco-stabilizing compound is present in an amount of from about 5 to about20 per cent.

16. The composition of claim 15 wherein from about 4 to about 10 percentof a detergent selected from the group consisting of nonionic andzwitterionic detergents is present.

17. The composition of claim 16 wherein the detergent is selected fromthe group consisting of condensation products of 1 mole of aliphaticalcohol having from eight to 22 carbon atoms with from 5 to 40 moles ofethylene oxide; 3.-(N,N-dimethyl-N-alkylammonio) propanel-sulfonatewherein the alkyl has from eight to 22 carbon atoms; and 3-(N,N-dimethyl-N- alkylammonio)-2-hydroxypropanel-sulfonate wherein thealkyl has from eight to 22 carbon atoms.

18. The composition of claim 17 wherein the detergent is3-(N,N-dimethyl-N-coconutalkylammonio)-2- hydroxypropanel -sulfonate.

19. The composition of claim 17 wherein the calcium salt is selectedfrom the group consisting of calcium acetate, calcium sulfate andcalcium choride'.

$3 3? UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,819,528 Dated June 25, 1974 Jim S. Berr Inventor-(s) y It is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Column 2, line 5, after invention delete "re" and insert therefor areColumn 3, line 34, after-"3" insert Colunn7, line 4, after "3-hyd'roxy"delete "tridecl" and insert therefor tridecyl Table II, first line,Column "A", insert 5 Table II, first line, Column "E" insert a hyphenTable II, first line, Column "4" delete "88" and insert therefor 100 aTable II, first line, Column "6", delete "100" and insert therefor 88Table II, line 2, Column "E", delete "9" Signed and Scaled this ninthD3) of December 1975 '[SEAL] Attest:

RUTH C. MA SON C. MARSHALL DANN Arresting Offzcer Commissioner ufPatentsand Trademarks $3 3? UNITED STATES PATENT OFFICE CERTIFICATE OFCORRECTION Patent No. 3,819,528 Dated June 25, 1974 Jim S. BerrInventor-(s) y It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 2, line 5, after invention delete "re" and insert therefor areColumn 3, line 34, after-"3" insert Colunn7, line 4, after "3-hyd'roxy"delete "tridecl" and insert therefor tridecyl Table II, first line,Column "A", insert 5 Table II, first line, Column "E" insert a hyphenTable II, first line, Column "4" delete "88" and insert therefor 100 aTable II, first line, Column "6", delete "100" and insert therefor 88Table II, line 2, Column "E", delete "9" Signed and Scaled this ninthD3) of December 1975 '[SEAL] Attest:

RUTH C. MA SON C. MARSHALL DANN Arresting Offzcer Commissioner ufPatentsand Trademarks

2. The composition of claim 1 wherein the co-stabilizing compound is analiphatic alcohol having the formula HO(CH2CH2O)xH wherein x is fromabout 2 to about 200; and wherein the amylolytic enzyme is an Alpha-amylase characterized by amylolytic activity in the pH range of fromabout 4.5 to about 10 and at a temperature of from about 60*F. to about150*F.
 2. from about 0.001 percent to about 1 percent amylolytic enzyme;2. from about 0.001 to about 1 percent amylolytic enzyme;
 3. from about0.001 to about 1 percent with respect to caclium ion of a water-soluble,enzyme-stabilizing calcium salt;
 3. from about 0.001 percent to about 1percent with respect to calcium ion of a water-solubleenzyme-stabilizing calcium salt;
 3. The composition of claim 2 whereinthe Alpha -amylase is derived from Bacillus subtilis.
 4. The compositionof claim 3 wherein the Alpha -amylase is present in an amount of about0.01 to about 0.5 percent.
 4. from about 2 percent to about 27 percentof an organic co-stabilizing compound selected from the group consistingof aliphatic glycols having the formula HO(CH2CH2O)xH wherein x is fromabout 2 to about 200; and 1,3-propanediol; and
 4. from about 2 to about27 percent of a 1,3-propanediol co-stabilizing compound; and
 5. from 0to about 15 percent of a detergent selected from the group consisting ofnonionic detergents and zwitterionic detergents.
 5. from 0 to about 15 %of a detergent selected from the group consisting of nonionic detergentsand zwitterionic detergents.
 5. The composition of claim 4 wherein thecalcium ion is present in an amount of about 0.005 to 0.05 percent andthe organic co-stabilizing compound is present in an amount of fromabout 5 percent to about 20 percent.
 6. The composition of claim 5wherein the organic costabilizing compound is selected from the groupconsisting of diethylene glycol; triethylene glycol; and glycols of theformula HO(CH2CH2O)xH wherein the average value of x is from 4 to about80.
 7. The composition of claim 6 wherein from about 4 to about 10percent of a nonionic or zwitterionic detergent is present.
 8. Thecomposition of claim 7 wherein the detergent is selected from the groupconsisting of condensation products of 1 mole of aliphatic alcoholhaving from eight to 22 carbon atoms with from 5 to 40 moles of ethyleneoxide; 3-(N,N-dimethyl-N-alkylammonio) propane-1-sulfonate wherein thealkyl has from eight to 22 carbon atoms; and3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-1-sulfonate wherein thealkyl has from eight to 22 carbon atoms.
 9. The composition of claim 8wherein the detergent is3-(N,N-dimethyl-N-coconutalkylammonio)-2-hydroxypropane-1-sulfonate andthe organic co-stabilizing compound is triethylene glycol.
 10. Thecomposition of claim 8 wherein the calcium salt is selected from thegroup consisting of calcium acetate, calcium sulfate and calciumchloride.
 11. A stabilized aqueous enzyme composition consistingessentially of by weight of the composition:
 12. The composition ofclaim 11 wherein the amylolytic enzyme is an Alpha -amylaseCharacterised by amylolytic activity in the pH range of from about 4.5to about 10 and at a temperature of from about 60*F. to about 150*F. 13.The Composition of claim 12 wherein the Alpha -amylase is derived fromBacillus subtilis.
 14. The composition of claim 13 wherein the Alpha-amylase is present in an amount of from about 0.01 to about 0.5percent.
 15. The composition of claim 14 wherein the calcium ion ispresent in an amount of from about 0.005 to 0.05 percent and the organicco-stabilizing compound is present in an amount of from about 5 to about20 percent.
 16. The composition of claim 15 wherein from about 4 toabout 10 percent of a detergent selected from the group consisting ofnonionic and zwitterionic detergents is present.
 17. The composition ofclaim 16 wherein the detergent is selected from the group consisting ofcondensation products of 1 mole of aliphatic alcohol having from eightto 22 carbon atoms with from 5 to 40 moles of ethylene oxide;3-(N,N-dimethyl-N-alkylammonio) propane-1-sulfonate wherein the alkylhas from eight to 22 carbon atoms; and3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-1-sulfonate wherein thealkyl has from eight to 22 carbon atoms.
 18. The composition of claim 17wherein the detergent is3-(N,N-dimethyl-N-coconutalkylammonio)-2-hydroxypropane-1-sulfonate. 19.The composition of claim 17 wherein the calcium salt is selected fromthe group consisting of calcium acetate, calcium sulfate and calciumchoride.